Induced pluripotent stem cells (iPSCs) offer unprecedented opportunities to advance our understanding and treatment of childhood diseases. One such disease is cystic fibrosis (CF). In the U.S., approximately 2,500 infants are born each year with CF and 30,000 children currently live with the disease. Many patients do not live past the age of 40. CF is caused by mutations in the CFTR gene, an anion channel, important in regulating electrolyte and water flow across mucus producing epithelia most notably the lung, pancreas and intestine. CF causing mutations cause abnormally viscous mucus in the airways, infection, inflammation, and lung destruction. Further complicating the situation is the number of cftr mutations, almost 2,000 described to date, and the variable disease severity. Determinants of this heterogeneity include the type of cftr mutation and both genetic and environmental modifiers5. The discovery of ivacaftor for treating the subset of cf patients (approx. 7%) with a G551D mutation was a significant advance in the field and represented the first mutation-targeted therapy6.
For most CF patients, including those with the most common mutation (F508del), effective treatments are not yet available. More potent CFTR correctors are needed. A major question for the community is how an individual's response to cftr modulators can be predicted? Children with CF will benefit the most from early, effective treatment. Some children with less common and less well-characterized mutations might respond to currently approved therapies. Identifying those patients is a priority. Clinical trials in this patient population and with rare mutations are challenging. Current data suggest that the future of effective CF treatment will be individually tailored combination therapy targeting distinct aspects of CFTR dysfunction7. These observations highlight the need for better surrogate preclinical models for drug development but ultimately as part of the routine care of individuals with CF.
A number of cell-based models of CF exist. High-throughput screens (HTS) using Fischer rat thyroid (FRT) cells led to the identification of a number of CFTR modulators. However, this platform is a poor predictor of clinical efficacy in part because thyroid cells are phenotypically different from airway epithelium and this “off the shelf” cell line does not carry the genetic background of patients. As such, although FRT cells are amenable to a HTS approach, these cells are phenotypically different from airway epithelium and the do not represent the genetic background of human patients. Air-liquid interface (ALI) cultures of human bronchial epithelial (HBE) cells8 more closely resemble the human airway epithelium. However, there are a number of disadvantages to using a HBE CF model: (1) an invasive procedure is required to obtain HBEs, (2) the cell numbers are limiting, (3) HBEs represent only one tissue-type and (4) HBEs are not well suited to genetic engineering (5) Using chamber and patch-clamp assays lack to capacity to measure large numbers of conditions simultaneously.
Intestinal organoid asssays22 have an established role in CF. The disadvantages to using intestinal organoids are (1) an intestinal or rectal biopsy is required, (2) although the cells can be cultured for long periods, they are not as well less suited to medium to high throughput approaches as iPSCs, and (3) they represent a different tissue (intestine) than the main tissue of interest in CF (lung).
Recent data suggests a promising role for iPSC in the study of CF9,10. iPSCs can be routinely and noninvasively generated from any patient, contain that individual's unique genetic background11. These cells can be expanded in culture to provide an inexhaustible supply of autologous cells. iPSCs are also suitable for gene-editing approaches10,12. Other groups have published modeling CF using iPSCs (9,18).
Directed differentiation of functional lung epithelial cell types from human pluripotent stem cells (PSCs) holds promise for in vitro modeling of complex respiratory diseases and for future cell-based regenerative therapies. Recent studies have demonstrated that a heterogeneous mixture of diverse lung epithelia accompanied by contaminating non-lung lineages can be simultaneously “co-derived” from PSCs differentiated in vitro (Dye et al., 2015; Firth et al., 2014; Gotoh et al., 2014; Green et al., 2011; Huang et al., 2013; Konishi et al., 2016; Longmire et al., 2012; Mou et al., 2012; Wong et al., 2012; Hawkins et al., 2017 and McCauley et al., 2017). However, many pulmonary diseases, such as cystic fibrosis, have their primary effects within distinct regions of the lungs and their constituent cellular subtypes. The heterogeneity of current differentiation outcomes therefore significantly hampers attempts to apply these PSC-based models to recapitulate pulmonary disease and test therapies in vitro. While recent cell sorting methods have enabled the derivation of more homogenous populations of early or primordial lung epithelial progenitor cells from human PSCs (hPSCs), Hawkins et al., 2017), the consistent derivation of well-defined mature functional lineages from these progenitors for effective disease modeling has remained challenging. This is due in part to heterogeneous, stochastic, or chaotic subsequent differentiation of these progenitors in protocols that are often reliant on many weeks or months of cell culture.
One approach to realize the promise of hPSC model systems for studying diseases affecting specific cellular subtypes is to engineer in vitro methods that more closely mimic in vivo developmental cell fate decisions. In contrast to current prolonged in vitro approaches, in vivo lung development is a tightly controlled process, where chaotic heterogeneity is minimized by signaling cascades that act cyclically in a regiospecific manner during narrow stage-dependent windows of time to precisely and rapidly promote appropriate cell fates while suppressing alternate fate options (Perrimon et al., 2012). The patterning of early lung epithelial progenitors in vivo is a classic example of this phenomenon, since soon after lineage specification of primordial lung epithelial progenitors, indicated by emergence of Nkx2-1+ endoderm, their descendants located at advancing distal lung bud tips are iteratively faced with the fate option of either maintaining a distal phenotype or surrendering this fate to assume a proximal airway cell fate (Rawlins et al., 2009). Through these repeated fate decisions, the branching lung airways are patterned post-specification along a proximodistal axis, which is canonically defined by the expression of key transcription factors SOX2 in the proximal developing airway and tracheal epithelium and SOX9 in the budding distal tips(Liu and Hogan, 2002; Que et al., 2009)
Recreating this tightly controlled proximodistal patterning of lung cells during in vitro differentiation of iPSC-derived NKX2-1+ progenitors has been difficult in part due to the plethora of developmental signaling pathways that have been described in mouse models as being important to this process, including Wnt, FGF, BMP, TGFβ, RA, SHUT, and Notch signaling (Bellusci et al., 1997; Cardoso et al., 1997; Chen et al., 2010; 2007; Hashimoto et al., 2012; Hyatt et al., 2004; Mucenski et al., 2003; Sekine et al., 1999; Shu et al., 2005; Y. Wang et al., 2013; Weaver et al., 2000; 1999; Zemke et al., 2009; Zhou et al., 1996). In particular, it has been noted that these pathways exhibit high levels of temporal and regional specificity by which they each promote the migration, differentiation, and maturation of specific cell types at the expense of others.
Perturbations to airway epithelial cell fate are involved in the pathology of many common and incurable pulmonary diseases yet the pathways involved in normal lung cell fate specification remain poorly understood. There is therefore a critical need for studies of the mechanisms by which temporal and spatial control of cell signaling leads to the development of specific lung lineages. The directed differentiation of human lung progenitors from pluripotent stem cells (hPSCs) is a potential source of transient developmental progenitors for these high-resolution studies. Importantly, there remains a critical lack of protocols for deriving airway progenitors from human pluripotent stem cells. Accordingly, there is an urgent need for a rapid, reliable and simple method for producing human airway epithelial cells from iPSC or human iPSCs that does not result in a heterogeneous population of cells.
Additionally, CF is the most common genetic lung disease and second only to sickle cell anemia as a life-shortening, genetic disease, and is caused by mutations in the CFTR gene. There is a pressing need for scalable, human platforms to predict an individual's response to existing CF therapies and to identify novel compounds.